Polynucleotide Encoding a Maize Herbicide Resistance Gene and Methods for Use

ABSTRACT

This invention relates to polynucleotide sequences encoding a gene that can confer resistance to at least one herbicide. It further relates to plants and seeds of plants carrying chimeric genes comprising said polynucleotide sequences, which enhance or confer resistance to at least one herbicide, and methods of making said plants and seeds. The invention further presents sequences that can be used as molecular markers that in turn can be used to identify the region of interest in corn lines resulting from new crosses and to quickly and efficiently select the best lines for breeding strategies by avoiding sensitive lines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Utility applicationSer. No. 11/683,737, filed Mar. 8, 2007, which claims the benefit ofU.S. Provisional Application Ser. No. 60/780,946, filed Mar. 9, 2006 andU.S. Provisional Application Ser. No. 60/888,634 filed Feb. 7, 2007, thecontents of which are herein incorporated by reference in theirentirety.

FIELD OF THE INVENTION

This invention relates to compositions and methods useful in creating orenhancing herbicide resistance in plants. Additionally, the inventionrelates to plants that have been genetically transformed with thecompositions of the invention.

BACKGROUND OF THE INVENTION

In the commercial production of crops, it is desirable to easily andquickly eliminate unwanted plants (i.e., “weeds”) from a field of cropplants. An ideal treatment would be one which could be applied to anentire field but which would eliminate only the unwanted plants whileleaving the crop plants unharmed. One such treatment system involves theuse of crop plants that are tolerant to a herbicide. When the herbicideis sprayed on a field of herbicide-tolerant crop plants, the crop plantscontinue to thrive while non-herbicide-tolerant weeds are killed orseverely damaged.

Crop tolerance to specific herbicides can be conferred by engineeringgenes into crops which encode appropriate herbicide metabolizingenzymes. In some cases these enzymes, and the nucleic acids that encodethem, originate in a plant. In other cases, they are derived from otherorganisms, such as microbes. See, e.g., Padgette et al. (1996) “New weedcontrol opportunities: Development of soybeans with a Round UP Ready™gene” and Vasil (1996) “Phosphinothricin-resistant crops,” both inHerbicide-Resistant Crops, ed. Duke (CRC Press, Boca Raton, Fla.) pp.54-84 and pp. 85-91. Indeed, transgenic plants have been engineered toexpress a variety of herbicide tolerance genes from a variety oforganisms, including a gene encoding a chimeric protein of ratcytochrome P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase(Shiota et al. (1994) Plant Physiol. 106: 17), among other plant P450genes (see, for example, Didierjean, L. et al. (2002) Plant Physiol.130: 179-189; Morant, M. S. et al. (2003) Opinion in Biotechnology14:151-162). Other genes that confer tolerance to herbicides include:acetohydroxy acid synthase (“AHAS”), which has been found to conferresistance to multiple types of ALS herbicides on plants expressing itand has been introduced into a variety of plants (see, e.g., Hattori etal. (1995) Mol. Gen. Genet. 246: 419); glutathione reductase andsuperoxide dismutase (Aono et al. (1995) Plant Cell Physiol. 36: 1687);and genes for various phosphotransferases (Datta et al. (1992) PlantMol. Biol. 20: 619).

While herbicide-tolerant crop plants are presently commerciallyavailable, improvements in every aspect of crop production arecontinuously in demand. Herbicides and crops that are presentlycommercially available unfortunately have particular characteristicswhich can limit their usefulness in commercial crop production.Particularly, individual herbicides have different and incompletespectra of activity against common weed species.

The acetolactate synthase, or ALS (also known as AHAS) family ofherbicides control weeds by inhibiting the production of branch chain ofamino acids that are essential to plant growth and development.Specifically, they bind to the plant ALS enzyme. Commonly usedherbicides in this family include nicosulfuron, rimsulfuron, andchlorsulfuron, among others. Herbicides in this category can be quitecrop-specific. Embodiments of the invention relate to plants that areresistant to members of the ALS-inhibiting class of herbicides, whichencompasses 5 sub-classes of herbicides including, but not limited to,the sulfonylurea (SU) family of herbicides and the imidazolinone familyof herbicides.

The pigment synthesis-inhibiting class of herbicides targets the enzymesthat allow plants to synthesize pigments, such as carotenoid pigments orchlorophyll pigments. Loss of pigment results in photo-destruction ofchlorophyll and whitening of plant tissues, which is why theseherbicides are often called “bleaching” herbicides. An example of asub-class of the bleaching herbicides is the HPPD-inhibiting class,which inhibits the 4-hydroxyphenylpyruvate dioxygenase (HPPD) enzyme(Lee et al. (1997) Weed Sci. 45:601-609). Herbicides in this familyinclude, but are not limited to, mesotrione, tembotrione, topramezoneand sulcotrione, among others. Corn is generally tolerant to mesotrionedue to metabolism of the herbicide (Mitchell et al. (2001) Pest Mgt.Sci. 57:120-128). The same detoxification system may give tolerance toboth mesotrione and some SU herbicides (Green & Williams (2004)Proceedings Weed Science Society of America 44:13). Embodiments of theinvention relate to plants that are resistant to members of the pigmentsynthesis-inhibiting class of herbicides.

The protoporphyrinogen oxidase (PPO)-inhibiting class of herbicidesinterferes with the synthesis of chlorophyll, resulting in compoundsthat produce highly active compounds (free-radicals). These reactivecompounds disrupt cell membranes which results in the leaf burningassociated with post-emergence applications of these products.Herbicides in this family include, but are not limited to, acifluorfen,fomesafen, lactofen, sulfentrazone, carfentrazone, flumiclorac andflumioxazin, among others. Embodiments of the invention relate to plantsthat are resistant to members of the PPO-inhibiting class of herbicides.

Photosystem II (PSII)-inhibiting herbicides have a mode of action thatinvolves interaction with components in the electron transfer chain ofPhotosystem II. Photosynthesis requires the transfer of electrons fromPhotosystem II to Photosystem I. A key step in this electron transferchain is the reduction of plastoquinone (PQ) by the D₁ protein in thethylakoid membrane. PSII-inhibitor herbicides bind to the D₁ protein,thus inhibiting PQ binding and interrupting the electron transferprocess. This results in the plants not being able to fix carbon dioxideand produce the carbohydrates needed for the plant to survive. The blockin electron transfer also causes an oxidative stress and the generationof radicals which cause rapid cellular damage. PSII-inhibitingherbicides are represented by several herbicide families, including thesymmetrical triazines, triazinones (asymmetrical triazines), substitutedureas, uracils, pyridazinones, phenyl carbamates, nitrites,benzothiadiazoles, phenyl pyridazines, and acid amides. Embodiments ofthe invention relate to plants that are resistant to members of the PSII-inhibiting class of herbicides.

Synthetic auxin herbicides are a widely used class of herbicides thatmimic the natural auxin hormones produced by plants. Auxins regulatemany plant processes, including cell growth and differentiation. Auxinsare generally present at low concentrations in the plant. Syntheticauxin herbicides mimic natural auxins and cause relatively highconcentrations in the cell that result in a rapid growth response.Susceptible plants treated with these herbicides exhibit symptoms thatcould be called ‘auxin overdose’, and eventually die as a result ofincreased rates of disorganized and uncontrolled growth. Embodiments ofthe invention relate to plants that are resistant to members of thesynthetic auxin class of herbicides.

Some embodiments of this invention are based on the fine mapping,cloning and characterization of the gene responsible for an importantherbicide resistance mechanism in maize.

It has been known since the early 1990s that natural tolerance in maize(Zea mays L.) to a subset of sulfonylurea herbicides (nicosulfuron[Dupont Accent® herbicide], rimsulfuron, primisulfuron, andthifensulfuron) is controlled by a single gene (named nsf by Kang (1993)Journal of Heredity 84(3): 216-217), with resistance dominant andsensitivity recessive (Harms et al. (1990) Theor. Appl. Genet.80:353-358; Kang (1993) supra; Green & Uhlrich (1993) Weed Sci.41:508-516; Green & Uhlrich (1994) Pestic. Sci. 40:187-191). It is alsoknown that tolerant maize plants metabolize nicosulfuron byhydroxylation, with the characteristics of a cytochrome P450(Fonne-Pfister et al. (1990) Pesticide Biochem. Physiol. 37:165-173;Brown & Cotterman (1994) Chem. Plant Prot. 10:47-81). It has beensuggested that the same corn gene responsible for determining toleranceto some sulfonylurea herbicides is also responsible for the tolerance tobentazon (Barrett et al. (1997) Role of cytochrome P-450 in herbicidemetabolism and selectivity and multiple herbicide metabolizingcytochrome P-450 activities in maize. In K. K. Hatzios, ed. Regulationof Enzymatic Systems Detoxifying Xenobiotics in Plants. Dordrecht:Kluwer Academic. pp. 35-50; Green (1998) Weed Technology 12:474-477) andHPPD inhibitor herbicides such as mesotrione (Green & Williams (2004)supra; Williams et al. (2005) HortScience 40(6):1801-1805). Recentadvances in the development of the maize physical map and integratedmarkers (Bortiri et al. (2006) Curr Opin Plant Biol. 9(2):164-71) hasallowed a positional cloning approach to be used for identifying theNsf1 locus.

The Nsf1 resistance gene of the embodiments of the present inventionencodes a novel gene related to the cytochrome P450 family. Whilemultiple cytochrome P450 genes have been described, they differ widelyin their response to different pathogens and exact action. The novelcytochrome P450 gene described in this disclosure has been demonstratedto provide improved tolerance or resistance to numerous herbicides,including nicosulfuron, rimsulfuron, primisulfuron, thifensulfuron andmesotrione.

SUMMARY OF THE INVENTION

The present invention is directed to embodiments including an isolatedpolynucleotide comprising a nucleotide sequence encoding a polypeptidecapable of conferring resistance to at least one herbicide, wherein thepolypeptide has an amino acid sequence of at least 85, 90 or 95%identity, when compared to SEQ ID NO:1 based on the Needleman-Wunschalignment algorithm, or a complement of the nucleotide sequence, whereinthe complement and the nucleotide sequence consist of the same number ofnucleotides and are 100% complementary. The herbicides to which thepolynucleotide of the embodiments imparts resistance include members ofthe ALS-inhibiting class; the pigment synthesis-inhibiting class; thePPO-inhibiting class; the PS II-inhibiting class; and the syntheticauxin class of herbicides. The polynucleotide of the embodiments mayimpart resistance to one or more herbicides from the same class, or fromdifferent classes, including representative members from all 5 classes.

Additional embodiments of the present invention include a vectorcomprising the polynucleotide of the embodiments and a recombinant DNAconstruct comprising the polynucleotide of the embodiments, operablylinked to at least one regulatory sequence. A plant cell, as well as aplant and a seed each comprising the recombinant DNA construct of anembodiment of the present invention are also encompassed. Also includedare plants comprising additional polynucleotides encoding polypeptidesresponsible for traits of interest, such as polypeptides havingglyphosate N-acetyltransferase activity, insecticidal Bt polypeptides,and other polypeptides of interest. Plants comprising thesepolynucleotides include monocots and dicots, including, but not limitedto, maize, wheat, barley, oats, switchgrass, sorghum, rice, soybean,canola, potato, cotton, and sunflower.

The methods embodied by the present invention include 1) a method fortransforming a cell, comprising transforming a cell with thepolynucleotide of an embodiment of the present invention, 2) a methodfor producing a plant comprising transforming a plant cell with therecombinant DNA construct of an embodiment of the present invention andregenerating a plant from the transformed plant cell, and 3) methods ofconferring or enhancing resistance to at least one herbicide, comprisingtransforming a plant with the recombinant DNA construct of an embodimentof the present invention, thereby conferring or enhancing resistance toat least one herbicide, such as a member of the ALS-inhibiting class;the pigment synthesis-inhibiting class; the PPO-inhibiting class; the PSII-inhibiting class; and the synthetic auxin class of herbicides.

In addition, an embodiment of the invention is a variant allele of theNsf1 sequence in which a specific single amino acid change (see Example2) renders the gene inoperative, resulting in sensitivity to at leastone ALS or HPPD inhibitor herbicide to which most corn is resistant.Accordingly, an additional method embodied by the present invention is amethod of using the variant of the Nsf1 gene as a marker in breedingstrategies to avoid incorporating the sensitive allele.

Methods of altering the level of expression of a protein capable ofconferring resistance to at least one herbicide in a plant cellcomprising (a) transforming a plant cell with the recombinant DNAconstruct of an embodiment of the present invention and (b) growing thetransformed plant cell under conditions that are suitable for expressionof the recombinant DNA construct wherein expression of the recombinantDNA construct results in production of altered levels of a proteincapable of conferring resistance to at least one herbicide in thetransformed host are also embodied by the present invention. Theherbicides for which resistance may be conferred include, for example,members of the ALS-inhibiting class; the pigment synthesis-inhibitingclass; the PPO-inhibiting class; the PS II-inhibiting class; and thesynthetic auxin class of herbicides.

Herbicides to which a polynucleotide of the embodiments may confer orenhance resistance include, but are not limited to, herbicides selectedfrom the ALS-inhibiting class of herbicides such as nicosulfuron,rimsulfuron, primisulfuron, imazethapyr, chlorsulfuron, chlorimuronethyl, triasulfuron, flumetsulam and imazaquin. Additionally, suchherbicides may be selected from the pigment synthesis-inhibiting classof herbicides, such as isoxaflutole, topramezone, sulcatrione andtembotrione. Such herbicides may also be selected from thePPO-inhibiting class of herbicides, such as acifluorfen, flumioxan andsulfentrazone. Optionally, such herbicides may be selected from the PSII-inhibiting class of herbicides, such as diuron, linuron, bentazon andchlorotoluron. Such herbicides may also be selected from the syntheticauxin class of herbicides, such as dicamba.

Methods of the embodiments include a method of determining the presenceof the polynucleotide of the embodiments or the Nsf1 locus in a plant,comprising at least one of: (a) isolating nucleic acid molecules fromthe plant and determining if an Nsf1 gene is present by attempting toamplify sequences homologous to the polynucleotide; or (b) isolatingnucleic acid molecules from the plant and performing a Southernhybridization, or (c) isolating proteins from the plant and performing awestern blot using antibodies to the NSF1 protein, or (d) isolatingproteins from the plant and performing an ELISA assay using antibodiesto the NSF1 protein, thereby determining the presence of thepolynucleotide of claim 1 in the plant.

Also encompassed by the embodiments are plants with enhanced toleranceto at least one herbicide, comprising the Nsf1 gene in a recombinant DNAconstruct. Such plants further comprise a second herbicide resistancegene providing a certain level of tolerance to a herbicide selected froma class of herbicides selected from the group consisting of:

-   -   (a) the ALS-inhibiting class;    -   (b) the pigment synthesis-inhibiting class;    -   (c) the PPO-inhibiting class;    -   (d) the PS II-inhibiting class; and    -   (e) the synthetic auxin class;        such that the presence of the Nsf1 gene confers upon the plant a        higher level of tolerance to the same herbicide than the        tolerance level exhibited by a plant comprising the second        herbicide resistance gene but not comprising the Nsf1 gene.

Also encompassed by the embodiments are soybean plants comprising theNsf1 gene, wherein such soybean plants also exhibit soybean cystnematode resistance. Such plants may have been created throughtransformation or plant breeding techniques, and may have been bred fromgermplasm such as those selected from the group consisting of, Peking,PI88788, PI89772, PI90763, PI209332, PI404189A, PI437654, PI438489B,PI467312, PI468916, Hartwig, J87-233, and progeny derived from any ofthe listed sources.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(a-e) is a multiple sequence alignment of the polypeptide sequenceof the embodiments (SEQ ID NO: 2) comparing it to other known CytochromeP450 polypeptides (SEQ ID NOs: 3-13). FIG. 1 d also indicates theposition of the most commonly conserved domain of the cytochrome P450family (SEQ ID NO: 14). Identical residues in the alignment areindicated in upper case letters.

FIG. 2(a-b) is a multiple sequence alignment of the polypeptidesequences of several sensitive and resistant corn lines showing thecommonly conserved domain of the cytochrome P450 family (SEQ ID NO: 14)as well as variations among the sequences.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention provide compositions and methodsdirected to inducing herbicide resistance in plants. The compositionsare novel nucleotide and amino acid sequences that confer or enhanceresistance to one or more members of one or more classes of herbicides,including the ALS-inhibiting, PPO-inhibiting, pigmentsynthesis-inhibiting, PS II-inhibiting and synthetic auxin herbicideclasses, whose members include, but are not limited to, nicosulfuron,rimsulfuron, primisulfuron, and mesotrione. Specifically, certainembodiments provide polypeptides having the amino acid sequence setforth in SEQ ID NO: 2, and variants and fragments thereof. Isolatednucleic acid molecules, and variants and fragments thereof, comprisingnucleotide sequences that encode the amino acid sequence shown in SEQ IDNO: 2 are further provided.

One example of the native nucleotide sequence that encodes thepolypeptide of SEQ ID NO: 2 is set forth in SEQ ID NO: 1. Plants, plantcells, seeds, and microorganisms comprising a nucleotide sequence thatencodes a polypeptide of the embodiments are also disclosed herein.

The full length polypeptide of the embodiments (SEQ ID NO: 2) sharesvarying degrees of homology with known polypeptides of the cytochromeP450 family. In particular, the novel polypeptide of the embodimentsshares homology with cytochrome P450 proteins isolated from Oryzasativa: Accession Nos. XP_(—)469850 (SEQ ID NO: 3), ABC69856 (SEQ ID NO:4); XP_(—)469849 (SEQ ID NO: 11) and XP_(—)469851 (SEQ ID NO: 12); andXP_(—)469852 (SEQ ID NO: 13) and Lolium rigidum: Accession Nos. AAK38080(SEQ ID NO: 5); AAK38079 (SEQ ID NO: 6); AAK38081 (SEQ ID NO: 7);BAD27508 (SEQ ID NO: 8); BAD27507 (SEQ ID NO: 9) and BAD27506 (SEQ IDNO: 10). FIG. 1 provides an alignment of the amino acid sequence setforth in SEQ ID NO: 2 with the O. sativa and L. rigidum cytochrome P450proteins (SEQ ID NOs: 3-13).

Amino acid alignments performed using the GAP program indicate that SEQID NO:2 shares the sequence similarities shown in Table 1 with the O.sativa and L. rigidum cytochrome P450 proteins. TABLE 1 Comparison ofNSF1 Peptide to other Cytochrome P450 peptides Other Cytochrome P450Protein Percent Identity Percent Similarity XP_469850 (SEQ ID NO: 3) 67%76% ABC69856 (SEQ ID NO: 4) 67% 76% AAK38080 (SEQ ID NO: 5) 68% 76%AAK38079 (SEQ ID NO: 6) 67% 77% AAK38081 (SEQ ID NO: 7) 67% 76% BAD27508(SEQ ID NO: 8) 67% 76% BAD27507 (SEQ ID NO: 9) 67% 76% BAD27506 (SEQ IDNO: 10) 67% 76% XP_469849 (SEQ ID NO: 11) 66% 75% XP_469851 (SEQ ID NO:12) 61% 71% XP_469852 (SEQ ID NO: 13) 60% 72%

The cytochrome P450 family of genes in plants catalyze extremely diverseand often complex regiospecific and/or stereospecific reactions in thebiosynthesis or catabolism of plant bioactive molecules. (Morant et al.(2003) Curr. Opin. Biotech. 14(2): 151-162). P450s are heme proteinsthat catalyze the activation of molecular oxygen by using electrons fromNADPH. In the Arabidopsis thaliana genome alone, there are an estimatedover 300 cytochromes P450 (Werck-Reichhart et al. (2000) Trends in PlantScience 5(3): 116-123). Common structural features occur in plantcytochromes P450 and help identify them as such. These features includethe F-X-X-G-X-R-X-C-X-G (SEQ ID NO: 14) motif generally found near theC-terminus (see FIG. 1 d). About 150 residues upstream, anotherconserved motif generally found follows the A/G-G-X-D/E-T-T/S (SEQ IDNO: 15) motif and corresponds to the region of the peptide responsiblefor oxygen-binding and activation.

The nucleic acids and polypeptides of the embodiments find use inmethods for conferring or enhancing herbicide resistance to a plant.Accordingly, the compositions and methods disclosed herein are useful inprotecting plants from damage caused by herbicides. “Herbicideresistance” is intended to mean that a plant or plant cell has theability to tolerate a higher concentration of a herbicide than plants orcells which are not resistant, or to tolerate a certain concentration ofa herbicide for a longer time than cells or plants which are notresistant. That is, herbicides are prevented from causing plant injury,or the injury caused by the herbicide is minimized or lessened, such as,for example, the reduction of leaf yellowing and associated yield loss.One of skill in the art will appreciate that the compositions andmethods disclosed herein can be used with other compositions and methodsavailable in the art for increasing or enhancing plant herbicideresistance. The term “enhance” refers to improve, increase, amplify,multiply, elevate, raise, and the like.

In particular aspects, the embodiments include methods for conferring orenhancing herbicide resistance in a plant comprising introducing into aplant at least one DNA construct, wherein the DNA construct comprises anucleotide sequence encoding a herbicide resistance polypeptide of theembodiments operably linked to a promoter that drives expression in theplant. The plant expresses the polypeptide, thereby conferring orenhancing herbicide resistance upon the plant, or improving the plant'sinherent level of resistance. In particular embodiments, the geneconfers or enhances resistance to at least one herbicide of theALS-inhibiting, pigment synthesis-inhibiting, PPO-inhibiting, PSII-inhibiting or synthetic auxin herbicide classes, whose membersinclude, but are not limited to, the herbicides nicosulfuron,rimsulfuron, primisulfuron, thifensulfuron, bentazon, and mesotrione.

Expression of a polypeptide of the embodiments may be targeted tospecific plant tissues, but generally in the case of herbicideresistance, continuous expression is desired throughout the cells of aplant. Therefore, while many promoters could be used in the embodimentsof the invention, generally constitutive promoters are utilized. Aconstitutive promoter is a promoter that directs expression of a genethroughout the various parts of a plant and continuously throughoutplant development.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues (e.g., peptide nucleic acids) having the essential nature ofnatural nucleotides in that they hybridize to single-stranded nucleicacids in a manner similar to naturally occurring nucleotides.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. Polypeptides of the embodiments can be produced either from anucleic acid disclosed herein, or by the use of standard molecularbiology techniques. For example, a truncated protein of the embodimentscan be produced by expression of a recombinant nucleic acid of theembodiments in an appropriate host cell, or alternatively by acombination of ex vivo procedures, such as protease digestion andpurification.

As used herein, the terms “encoding” or “encoded” when used in thecontext of a specified nucleic acid mean that the nucleic acid comprisesthe requisite information to direct translation of the nucleotidesequence into a specified protein. The information by which a protein isencoded is specified by the use of codons. A nucleic acid encoding aprotein may comprise non-translated sequences (e.g., introns) withintranslated regions of the nucleic acid or may lack such interveningnon-translated sequences (e.g., as in cDNA).

The embodiments of the invention encompass isolated or substantiallypurified polynucleotide or protein compositions. An “isolated” or“purified” polynucleotide or protein, or biologically active portionthereof, is substantially or essentially free from components thatnormally accompany or interact with the polynucleotide or protein asfound in its naturally occurring environment. Thus, an isolated orpurified polynucleotide or protein is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Optimally, an “isolated”polynucleotide is free of sequences (optimally protein encodingsequences) that naturally flank the polynucleotide (i.e., sequenceslocated at the 5′ and 3′ ends of the polynucleotide) in the genomic DNAof the organism from which the polynucleotide is derived. For example,in various embodiments, the isolated polynucleotide can contain lessthan about 5 kb, about 4 kb, about 3 kb, about 2 kb, about 1 kb, about0.5 kb, or about 0.1 kb of nucleotide sequence that naturally flank thepolynucleotide in genomic DNA of the cell from which the polynucleotideis derived. A protein that is substantially free of cellular materialincludes preparations of protein having less than about 30%, about 20%,about 10%, about 5%, or about 1% (by dry weight) of contaminatingprotein. When the protein of the embodiments, or a biologically activeportion thereof, is recombinantly produced, optimally culture mediumrepresents less than about 30%, about 20%, about 10%, about 5%, or about1% (by dry weight) of chemical precursors or non-protein-of-interestchemicals.

Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the embodiments. By“fragment” is intended a portion of the nucleotide sequence or a portionof the amino acid sequence and hence protein encoded thereby. Fragmentsof a nucleotide sequence may encode protein fragments that retain thebiological activity of the native protein and hence have the ability toconfer or enhance resistance to at least one herbicide of theALS-inhibiting, PPO-inhibiting, pigment synthesis-inhibiting, PSII-inhibiting or synthetic auxin herbicide class. Alternatively,fragments of a nucleotide sequence that are useful as hybridizationprobes do not necessarily encode fragment proteins retaining biologicalactivity. Thus, fragments of a nucleotide sequence may range from atleast about 15 nucleotides, about 50 nucleotides, about 100 nucleotides,and up to the full-length nucleotide sequence encoding the polypeptidesof the embodiments.

A fragment of a nucleotide sequence that encodes a biologically activeportion of a polypeptide of the embodiments will encode at least about15, about 25, about 30, about 40, or about 50 contiguous amino acids, orup to the total number of amino acids present in a full-lengthpolypeptide of the embodiments (for example, 521 amino acids for SEQ IDNO: 2). Fragments of a nucleotide sequence that are useful ashybridization probes or PCR primers generally need not encode abiologically active portion of a protein.

As used herein, “full-length sequence” in reference to a specifiedpolynucleotide means having the entire nucleic acid sequence of a nativesequence. By “native sequence” is intended an endogenous sequence, i.e.,a non-engineered sequence found in an organism's genome.

Thus, a fragment of a nucleotide sequence of the embodiments may encodea biologically active portion of a polypeptide, or it may be a fragmentthat can be used as a hybridization probe or PCR primer using methodsdisclosed below. A biologically active portion of an herbicideresistance polypeptide can be prepared by isolating a portion of one ofthe nucleotide sequences of the embodiments, expressing the encodedportion of the protein and assessing the ability of the encoded portionof the protein to confer or enhance herbicide resistance in a plant.Nucleic acid molecules that are fragments of a nucleotide sequence ofthe embodiments comprise at least about 15, about 20, about 50, about75, about 100, or about 150 nucleotides, or up to the number ofnucleotides present in a full-length nucleotide sequence disclosedherein (for example, 1563 nucleotides for SEQ ID NO: 1).

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more internal sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotidesequence or amino acid sequence, respectively. One of skill in the artwill recognize that variants of the nucleic acids of the embodimentswill be constructed such that the open reading frame is maintained. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the polypeptides of the embodiments. Naturallyoccurring allelic variants such as these can be identified with the useof well-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant polynucleotides also include synthetically derivedpolynucleotide, such as those generated, for example, by usingsite-directed mutagenesis but which still encode a protein of theembodiments. Generally, variants of a particular polynucleotide of theembodiments will have at least about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99% or more sequence identity to thatparticular polynucleotide as determined by sequence alignment programsand parameters described elsewhere herein.

Variants of a particular polynucleotide of the embodiments (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Thus, for example, isolated polynucleotides that encodea polypeptide with a given percent sequence identity to the polypeptideof SEQ ID NO: 2 are disclosed. Percent sequence identity between any twopolypeptides can be calculated using sequence alignment programs andparameters described elsewhere herein. Where any given pair ofpolynucleotides of the embodiments is evaluated by comparison of thepercent sequence identity shared by the two polypeptides they encode,the percent sequence identity between the two encoded polypeptides is atleast about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99% or more sequence identity.

“Variant” protein is intended to mean a protein derived from the nativeprotein by deletion or addition of one or more amino acids at one ormore internal sites in the native protein and/or substitution of one ormore amino acids at one or more sites in the native protein. Variantproteins encompassed by the embodiments are biologically active, that isthey continue to possess the desired biological activity of the nativeprotein, that is, the ability to confer or enhance plant herbicideresistance as described herein. Such variants may result from, forexample, genetic polymorphism or from human manipulation. Biologicallyactive variants of a native protein of the embodiments will have atleast about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99% or more sequence identity to the amino acid sequence for thenative protein as determined by sequence alignment programs andparameters described elsewhere herein. A biologically active variant ofa protein of the embodiments may differ from that protein by as few asabout 1-15 amino acid residues, as few as about 1-10, such as about6-10, as few as about 5, as few as 4, 3, 2, or even 1 amino acidresidue.

The proteins of the embodiments may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants and fragments of the herbicideresistance proteins can be prepared by mutations in the DNA. Methods formutagenesis and polynucleotide alterations are well known in the art.See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492;Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No.4,873,192; Walker and Gaastra, eds. (1983) Techniques in MolecularBiology (MacMillan Publishing Company, New York) and the referencescited therein. Guidance as to appropriate amino acid substitutions thatdo not affect biological activity of the protein of interest may befound in the model of Dayhoff et al. (1978) Atlas of Protein Sequenceand Structure (Natl. Biomed. Res. Found., Washington, D.C.), hereinincorporated by reference. Conservative substitutions, such asexchanging one amino acid with another having similar properties, may beoptimal.

Thus, the genes and polynucleotides of the embodiments include both thenaturally occurring sequences as well as mutant forms. Likewise, theproteins of the embodiments encompass both naturally occurring proteinsas well as variations and modified forms thereof. Such variants willcontinue to possess the desired ability to confer or enhance plantresistance to at least one herbicide of the ALS-inhibiting,PPO-inhibiting, pigment synthesis-inhibiting, PS II-inhibiting orsynthetic auxin herbicide classes. Obviously, the mutations that will bemade in the DNA encoding the variant must not place the sequence out ofreading frame and optimally will not create complementary regions thatcould produce secondary mRNA structure. See, EP Patent No. 0075444.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by screening transgenic plants which have been transformedwith the variant protein to ascertain the effect on the herbicideresistance characteristics of the plant.

Variant polynucleotides and proteins also encompass sequences andproteins derived from mutagenic or recombinogenic procedures, includingand not limited to procedures such as DNA shuffling. One of skill in theart could envision modifications that would alter the range ofherbicides to which the protein responds. With such a procedure, one ormore different protein coding sequences can be manipulated to create anew protein possessing the desired properties. In this manner, librariesof recombinant polynucleotides are generated from a population ofrelated sequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between the protein geneof the embodiments and other known protein genes to obtain a new genecoding for a protein with an improved property of interest, such asincreased ability to confer or enhance plant herbicide resistance.Strategies for such DNA shuffling are known in the art. See, forexample, US 2002/0058249; Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997)Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

The polynucleotides of the embodiments can be used to isolatecorresponding sequences from other organisms, particularly other plants.In this manner, methods such as PCR, hybridization, and the like can beused to identify such sequences based on their sequence homology to thesequences set forth herein. Sequences isolated based on their sequenceidentity to the entire sequences set forth herein or to variants andfragments thereof are encompassed by the embodiments. Such sequencesinclude sequences that are orthologs of the disclosed sequences.“Orthologs” is intended to mean genes derived from a common ancestralgene and which are found in different species as a result of speciation.Genes found in different species are considered orthologs when theirnucleotide sequences and/or their encoded protein sequences share atleast about 60%, about 70%, about 75%, about 80%, about 85%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, or greater sequence identity. Functions oforthologs are often highly conserved among species. Thus, isolatedpolynucleotides that encode for a protein that confers or enhances plantherbicide resistance and that hybridize under stringent conditions tothe sequences disclosed herein, or to variants or fragments thereof, areencompassed by the embodiments.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, and are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

In hybridization techniques, all or part of a known polynucleotide isused as a probe that selectively hybridizes to other correspondingpolynucleotides present in a population of cloned genomic DNA fragmentsor cDNA fragments (i.e., genomic or cDNA libraries) from a chosenorganism. The hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker.Thus, for example, probes for hybridization can be made by labelingsynthetic oligonucleotides based on the polynucleotides of theembodiments. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed in Sambrook et al. (1989) supra.

For example, an entire polynucleotide disclosed herein, or one or moreportions thereof, may be used as a probe capable of specificallyhybridizing to corresponding polynucleotides and messenger RNAs. Toachieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique and are optimally at leastabout 10 nucleotides in length, at least about 15 nucleotides in length,or at least about 20 nucleotides in length. Such probes may be used toamplify corresponding polynucleotides from a chosen organism by PCR.This technique may be used to isolate additional coding sequences from adesired organism or as a diagnostic assay to determine the presence ofcoding sequences in an organism. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Sambrook et al. (1989) supra.

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a final wash in 0.1×SSC at 60 to 65° C. for at least 30minutes. Optionally, wash buffers may comprise about 0.1% to about 1%SDS. Duration of hybridization is generally less than about 24 hours,usually about 4 to about 12 hours. The duration of the wash time will beat least a length of time sufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the thermal melting point (T_(m))can be approximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridization,and/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with ≧90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the T_(m) for thespecific sequence and its complement at a defined ionic strength and pH.However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the T_(m); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the T_(m); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe T_(m). Using the equation, hybridization and wash compositions, anddesired T_(m), those of ordinary skill will understand that variationsin the stringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis optimal to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York). See Sambrook et al. (1989)supra.

Various procedures can be used to check for the presence or absence of aparticular sequence of DNA, RNA, or a protein. These include, forexample, Southern blots, northern blots, western blots, and ELISAanalysis. Techniques such as these are well known to those of skill inthe art and many references exist which provide detailed protocols. Suchreferences include Sambrook et al. (1989) supra, and Crowther, J. R.(2001), The ELISA Guidebook, Humana Press, Totowa, N.J., USA.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides: (a) “referencesequence,” (b) “comparison window,” (c) “sequence identity,” and, (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twopolynucleotides. Generally, the comparison window is at least about 20contiguous nucleotides in length, and optionally can be about 30, about40, about 50, about 100, or longer. Those of skill in the art understandthat to avoid a high similarity to a reference sequence due to inclusionof gaps in the polynucleotide sequence a gap penalty is typicallyintroduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, and are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of theembodiments. BLAST protein searches can be performed with the BLASTXprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to a protein or polypeptide of the embodiments. To obtaingapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0)can be utilized as described in Altschul et al. (1997) Nucleic AcidsRes. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used toperform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. See www.ncbi.nlm.nih.gov. Alignment may also be performedmanually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using Gap Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using Gap Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0,1, 2, 3, 4, 5, 6, 7, 8,9,10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, and no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo polynucleotides or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The use of the term “polynucleotide” is not intended to limit theembodiments to polynucleotides comprising DNA. Those of ordinary skillin the art will recognize that polynucleotides can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the embodiments also encompass all forms of sequencesincluding, and not limited to, single-stranded forms, double-strandedforms, and the like.

Isolated polynucleotides of the present invention can be incorporatedinto recombinant DNA constructs capable of introduction into andreplication in a host cell. A “vector” may be such a construct thatincludes a replication system and sequences that are capable oftranscription and translation of a polypeptide-encoding sequence in agiven host cell. A number of vectors suitable for stable transfection ofplant cells or for the establishment of transgenic plants have beendescribed in, e.g., Pouwels et al, Cloning Vectors: A Laboratory Manual,1985, supp. 1987; Weissbach and Weissbach, Methods for Plant MolecularBiology, Academic Press, 1989; and Flevin et al., Plant MolecularBiology Manual, Kluwer Academic Publishers, 1990. Typically, plantexpression vectors include, for example, one or more cloned plant genesunder the transcriptional control of 5′ and 3′ regulatory sequences anda dominant selectable marker. Such plant expression vectors also cancontain a promoter regulatory region (e.g., a regulatory regioncontrolling inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific expression), atranscription initiation start site, a ribosome binding site, an RNAprocessing signal, a signal peptide sequence for targeted expression, atranscription termination site, and/or a polyadenylation signal.

The terms “recombinant construct,” “expression cassette,” “expressionconstruct,” “chimeric construct,” “construct,” “recombinant DNAconstruct,” “DNA construct” and “recombinant DNA fragment” are usedinterchangeably herein and are nucleic acid fragments. A recombinantconstruct comprises an artificial combination of nucleic acid fragments,including, and not limited to, regulatory and coding sequences that arenot found together in nature. For example, a recombinant DNA constructmay comprise regulatory sequences and coding sequences that are derivedfrom different sources, or regulatory sequences and coding sequencesderived from the same source and arranged in a manner different thanthat found in nature. Such construct may be used by itself or may beused in conjunction with a vector. If a vector is used then the choiceof vector is dependent upon the method that will be used to transformhost cells as is well known to those skilled in the art. For example, aplasmid vector can be used. The skilled artisan is well aware of thegenetic elements that must be present on the vector in order tosuccessfully transform, select and propagate host cells comprising anyof the isolated nucleic acid fragments of the invention. Screening toobtain lines displaying the desired expression level and pattern of thepolynucleotides or of the Nsf1 locus may be accomplished byamplification, Southern analysis of DNA, Northern analysis of mRNAexpression, immunoblotting analysis of protein expression, phenotypicanalysis, and the like.

The term “recombinant DNA construct” refers to a DNA construct assembledfrom nucleic acid fragments obtained from different sources. The typesand origins of the nucleic acid fragments may be very diverse.

In some embodiments, DNA constructs comprising a promoter operablylinked to a heterologous nucleotide sequence of the embodiments arefurther provided. The DNA constructs of the embodiments find use ingenerating transformed plants, plant cells, and microorganisms and inpracticing the methods for inducing ALS and HPPD inhibitor herbicideresistance disclosed herein. The DNA construct will include 5′ and 3′regulatory sequences operably linked to a polynucleotide of theembodiments. “Operably linked” is intended to mean a functional linkagebetween two or more elements. “Regulatory sequences” refer tonucleotides located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and which mayinfluence the transcription, RNA processing, stability, or translationof the associated coding sequence. Regulatory sequences may include, andare not limited to, promoters, translation leader sequences, introns,and polyadenylation recognition sequences. For example, an operablelinkage between a polynucleotide of interest and a regulatory sequence(a promoter, for example) is functional link that allows for expressionof the polynucleotide of interest. Operably linked elements may becontiguous or non-contiguous. When used to refer to the joining of twoprotein coding regions, operably linked is intended to mean that thecoding regions are in the same reading frame. The coding sequence mayadditionally contain a sequence used to target the protein to thechloroplast, the vacuole, the endoplasmic reticulum or to the outside ofthe cell. The cassette may additionally contain at least one additionalgene to be cotransformed into the organism. Alternatively, theadditional gene(s) can be provided on multiple DNA constructs. Such aDNA construct is provided with a plurality of restriction sites and/orrecombination sites for insertion of the polynucleotide that encodes aherbicide resistance polypeptide to be under the transcriptionalregulation of the regulatory regions. The DNA construct may additionallycontain selectable marker genes.

The DNA construct will include in the 5′-3′ direction of transcription,a transcriptional initiation region (i.e., a promoter), translationalinitiation region, a polynucleotide of the embodiments, a translationaltermination region and, optionally, a transcriptional termination regionfunctional in the host organism. The regulatory regions (i.e.,promoters, transcriptional regulatory regions, and translationaltermination regions) and/or the polynucleotide of the embodiments may benative/analogous to the host cell or to each other. Alternatively, theregulatory regions and/or the polynucleotide of the embodiments may beheterologous to the host cell or to each other. As used herein,“heterologous” in reference to a sequence is a sequence that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention. For example, a promoter operably linkedto a heterologous polynucleotide is from a species different from thespecies from which the polynucleotide was derived, or, if from thesame/analogous species, one or both are substantially modified fromtheir original form and/or genomic locus, or the promoter is not thenative promoter for the operably linked polynucleotide.

The optionally included termination region may be native with thetranscriptional initiation region, may be native with the operablylinked polynucleotide of interest, may be native with the plant host, ormay be derived from another source (i.e., foreign or heterologous) tothe promoter, the polynucleotide of interest, the host, or anycombination thereof. Convenient termination regions are available fromthe Ti-plasmid of A. tumefaciens, such as the octopine synthase andnopaline synthase termination regions. See also Guerineau et al. (1991)Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfaconet al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989)Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic AcidsRes. 15:9627-9639. In particular embodiments, the potato proteaseinhibitor II gene (PinII) terminator is used. See, for example, Keil etal. (1986) Nucl. Acids Res. 14:5641-5650; and An et al. (1989) PlantCell 1:115-122, herein incorporated by reference in their entirety.

A number of promoters can be used in the practice of the embodiments,including the native promoter of the polynucleotide sequence ofinterest. The promoters can be selected based on the desired outcome. Awide range of plant promoters are discussed in the recent review ofPotenza et al. (2004) In Vitro Cell Dev Biol—Plant 40:1-22, hereinincorporated by reference. For example, the nucleic acids can becombined with constitutive, tissue-preferred, pathogen-inducible, orother promoters for expression in plants. Such constitutive promotersinclude, for example, the core promoter of the Rsyn7 promoter and otherconstitutive promoters disclosed in WO 99/43838 and U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171);ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 andChristensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last etal. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Other constitutive promoters include, for example, U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; 5,608,142; and 6,177,611.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

DNA constructs may additionally contain 5′ leader sequences. Such leadersequences can act to enhance translation. Translation leaders are knownin the art and include: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus), and humanimmunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991)Nature 353:90-94); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) inMolecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); andmaize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991)Virology 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968. Other methods known to enhance translation can alsobe utilized, for example, introns, and the like.

In preparing the DNA construct, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

The DNA construct can also comprise a selectable marker gene for theselection of transformed cells. Selectable marker genes are utilized forthe selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. CellScience 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), andyellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al.(2004) J. Cell Science 117:943-54). For additional selectable markers,see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318;Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad.Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph. D. Thesis, University of Heidelberg; Reines et al. (1993) Proc.Natl. Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the embodiments.

The gene of the embodiments can be expressed as a transgene in order tomake plants resistant to at least one herbicide of the ALS-inhibiting,PPO-inhibiting, pigment synthesis-inhibiting, PS II-inhibiting orsynthetic auxin herbicide classes. Using the different promotersdescribed elsewhere in this disclosure, this will allow its expressionin a modulated form in different circumstances. One can also insert theentire gene, both native promoter and coding sequence, as a transgene.Finally, using the gene of the embodiments as a transgene will allowquick combination with other traits, such as insect or fungalresistance.

In certain embodiments the nucleic acid sequences of the embodiments canbe stacked with any combination of polynucleotide sequences of interest,which may be transgenic or non-transgenic, in order to create plantswith a desired phenotype. For example, the polynucleotides of theembodiments may be stacked with any other polynucleotides of theembodiments, or with other genes. The combinations generated can alsoinclude multiple copies of any one of the polynucleotides of interest.The polynucleotides of the embodiments can also be stacked with anyother gene or combination of genes to produce plants with a variety ofdesired trait combinations including and not limited to traits desirablefor animal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529);balanced amino acids (e.g. hordothionins (U.S. Pat. Nos. 5,990,389;5,885,801; 5,885,802; and 5,703,409); barley high lysine (Williamson etal. (1987) Eur. J. Biochem. 165:99-106; and WO 98/20122); and highmethionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261:6279;Kirihara et al. (1988) Gene 71:359; and Musumura et al. (1989) PlantMol. Biol. 12: 123)); increased digestibility (e.g., modified storageproteins (U.S. application Ser. No. 10/053,410, filed Nov. 7, 2001); andthioredoxins (U.S. application Ser. No. 10/005,429, filed Dec. 3,2001)), the disclosures of which are herein incorporated by reference.The polynucleotides of the embodiments can also be stacked with traitsdesirable for insect, disease or herbicide resistance (e.g., Bacillusthuringiensis toxin proteins (U.S. Pat. Nos. 5,366,892; 5,747,450;5,737,514; 5723,756; 5,593,881; Geiser et al (1986) Gene 48:109);lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825); fumonisindetoxification genes (U.S. Pat. No. 5,792,931); avirulence and diseaseresistance genes (Jones et al. (1994) Science 266:789; Martin et al.(1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089);acetolactate synthase (ALS) mutants that lead to herbicide resistancesuch as the S4 and/or Hra mutations (Lee et al., (1988) EMBO J.7(5):1241-1248), resistance to inhibitors of glutamine synthase such asphosphinothricin or basta (e.g., bar gene; De Block et al. (1987) EMBOJ. 6:2513-2518); HPPD genes that confer tolerance to HPPD inhibitingherbicides such as mesotrione or isoxaflutole (Matringe et al. (2005)Pest Management Science 61:269-276; Dufourmantel et al., (2007) PlantBiotech. J. 5:118-133; see also WO1997049816), genes for tolerance toPPO inhibiting herbicides (Li and Nicholl (2005) Pest Management Science61:277-285); synthetic auxin resistance genes (US patent application2005/014737 and Herman et al., (2005) J. Biol. Chem. 280: 24759-24767),and glyphosate resistance (epsps genes, gat genes such as thosedisclosed in U.S. Patent Application Publication US2004/0082770, alsoWO02/36782 and WO03/092360)); and traits desirable for processing orprocess products such as high oil (e.g., U.S. Pat. No. 6,232,529);modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No.5,952,544; WO 94/11516)); modified starches (e.g., ADPGpyrophosphorylases (AGPase), starch synthases (SS), starch branchingenzymes (SBE) and starch debranching enzymes (SDBE)); and polymers orbioplastics (e.g., U.S. Pat. No. 5.602,321; beta-ketothiolase,polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert etal. (1988) J. Bacteriol. 170:5837-5847) facilitate expression ofpolyhydroxyalkanoates (PHAs)), the disclosures of which are hereinincorporated by reference. One could also combine the polynucleotides ofthe embodiments with polynucleotides providing agronomic traits such asmale sterility (e.g., see U.S. Pat. No. 5.583,210), stalk strength,flowering time, yield improvement, or transformation technology traitssuch as cell cycle regulation or gene targeting (e.g. WO 99/61619; WO00/17364; WO 99/25821), the disclosures of which are herein incorporatedby reference.

These stacked combinations can be created by any method including andnot limited to cross breeding plants by any conventional or TopCross®methodology, or genetic transformation. If the traits are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference.

Further embodiments include plants obtainable by a method comprising:crossing a plant containing the Nsf1 gene as a first parent plant, witha different plant that lacks an Nsf1 gene as a second parent plant,thereby to obtain progeny comprising the Nsf1 gene of the first parent;and optionally further comprising one or more further breeding steps toobtain progeny of one or more further generations comprising the Nsf1gene of the first parent. Such embodied plants can include both inbredand hybrid plants. Seeds of such plants, including those seeds which arehomozygous and heterozygous for the Nsf1 gene, and methods of obtainingplant products resulting from the processing of those seeds are embodiedin the invention. Using such seed in food or feed or the production of acorn product, such as flour, meal and oil is also an embodiment of theinvention.

An “ancestral line” or “progenitor” is a parent line used as a source ofgenes, e.g., for the development of elite lines. “Progeny” are thedescendents of the ancestral line, and may be separated from theirancestors by many generations of breeding. An “elite line” or “elitevariety” is an agronomically superior line or variety that has resultedfrom many cycles of breeding and selection for superior agronomicperformance. Similarly, “elite germplasm” is an agronomically superiorgermplasm, typically derived from and/or capable of giving rise to aplant with superior agronomic performance, such as an existing or newlydeveloped elite line of corn or soybeans.

Also embodied in the invention is the use of molecular markers to movethe gene or transgene into elite lines using breeding techniques.Molecular markers can be used in a variety of plant breedingapplications (eg see Staub et al. (1996) Hortscience 31: 729-741;Tanksley (1983) Plant Molecular Biology Reporter. 1: 3-8). One of themain areas of interest is to increase the efficiency of backcrossing andintrogressing genes using marker-assisted selection (MAS). A molecularmarker that demonstrates linkage with a locus affecting a desiredphenotypic trait provides a useful tool for the selection of the traitin a plant population. This is particularly true where the phenotype ishard to assay, e.g. many disease resistance traits, or, occurs at a latestage in the plants development, e.g. seed characteristics. Since DNAmarker assays are less laborious, and take up less physical space, thanfield phenotyping, much larger populations can be assayed, increasingthe chances of finding a recombinant with the target segment from thedonor line moved to the recipient line. The closer the linkage, the moreuseful the marker, as recombination is less likely to occur between themarker and the gene causing the trait, which can result in falsepositives. Having flanking markers decreases the chances that falsepositive selection will occur as a double recombination event would beneeded. The ideal situation is to have a marker in the gene itself, sothat recombination can not occur between the marker and the gene. Such amarker is called a ‘perfect marker’.

Optionally, the nucleic acids of the embodiments may be targeted to thechloroplast for expression. In this manner, where the nucleic acid isnot directly inserted into the chloroplast, the expression cassette willadditionally contain a nucleic acid encoding a transit peptide to directthe gene product of interest to the chloroplasts. Such transit peptidesare known in the art. See, for example, Von Heijne et al. (1991) PlantMol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem.264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968;Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; andShah et al. (1986) Science 233:478-481.

Chloroplast targeting sequences are known in the art and include thechloroplast small subunit of ribulose-1,5-bisphosphate carboxylase(Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol.30:769-780; Schnell et al. (1991) J. Biol. Chem. 266(5):3335-3342);5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al.(1990) J. Bioenerg. Biomemb. 22(6):789-810); tryptophan synthase (Zhaoet al. (1995) J. Biol. Chem. 270(11):6081-6087); plastocyanin (Lawrenceet al. (1997) J. Biol. Chem. 272(33):20357-20363); chorismate synthase(Schmidt et al. (1993) J. Biol. Chem. 268(36):27447-27457); and thelight harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al.(1988) J. Biol. Chem. 263:14996-14999). See also Von Heijne et al.(1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol.Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol.84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun.196:1414-1421; and Shah et al. (1986) Science 233:478-481.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530;Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab andMaliga (1993) EMBO J. 12:601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

The nucleic acids to be targeted to the chloroplast may be optimized forexpression in the chloroplast to account for differences in codon usagebetween the plant nucleus and this organelle. In this manner, thenucleic acids of interest may be synthesized using chloroplast-preferredcodons. See, for example, U.S. Pat. No. 5,380,831, herein incorporatedby reference.

The methods of the embodiments may involve, and are not limited to,introducing a polypeptide or polynucleotide into a plant. “Introducing”is intended to mean presenting to the plant the polynucleotide. In someembodiments, the polynucleotide will be presented in such a manner thatthe sequence gains access to the interior of a cell of the plant,including its potential insertion into the genome of a plant. Themethods of the embodiments do not depend on a particular method forintroducing a sequence into a plant, only that the polynucleotide gainsaccess to the interior of at least one cell of the plant. Methods forintroducing polynucleotides into plants are known in the art including,and not limited to, stable transformation methods, transienttransformation methods, and virus-mediated methods.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. “Host cell” refersthe cell into which transformation of the recombinant DNA constructtakes place and may include a yeast cell, a bacterial cell, and a plantcell. Examples of methods of plant transformation includeAgrobacterium-mediated transformation (De Blaere et al., 1987, Meth.Enzymol. 143:277) and particle-accelerated or “gene gun” transformationtechnology (Klein et al, 1987, Nature (London) 327:70-73; U.S. Pat. No.4,945,050), among others.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” or “transient expression” is intended to meanthat a polynucleotide is introduced into the plant and does notintegrate into the genome of the plant or a polypeptide is introducedinto a plant.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (U.S. Pat. Nos. 5,563,055-and 5,981,840), direct genetransfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballisticparticle acceleration (see, for example, Sanford et al., U.S. Pat. Nos.4,945,050; 5,879,918; 5,886,244; and 5,932,782; Tomes et al. (1995) inPlant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborgand Phillips (Springer-Verlag, Berlin); McCabe et al. (1988)Biotechnology 6:923-926); and Lec1 transformation (WO 00/28058). Alsosee Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al.(1987) Particulate Science and Technology 5:27-37 (onion); Christou etal. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat.Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) PlantPhysiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman etal. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) PlantCell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992)Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant CellReports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide of the embodiments can be contained in transfercassette flanked by two non-identical recombination sites. The transfercassette is introduced into a plant have stably incorporated into itsgenome a target site which is flanked by two non-identical recombinationsites that correspond to the sites of the transfer cassette. Anappropriate recombinase is provided and the transfer cassette isintegrated at the target site. The polynucleotide of interest is therebyintegrated at a specific chromosomal position in the plant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the embodiments provides transformed seed (also referredto as “transgenic seed”) having a nucleotide construct of theembodiments, for example, a DNA construct of the embodiments, stablyincorporated into their genome.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which maize plant can be regenerated,plant calli, plant clumps, and plant cells that are intact in plants orparts of plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Grain is intended to mean the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included within the scope of theembodiments, provided that these parts comprise the introducedpolynucleotides.

The embodiments of the invention may be used to confer or enhanceherbicide resistance in plants, especially soy (Glycine max). Otherplant species may also be of interest in practicing the embodiments ofthe invention, including, and not limited to, other dicot and monocotcrop plants. The maize gene of the embodiments is commonly found in themajority of commercial corn lines, most of which are naturally tolerantto at least one, and usually several, synthetic auxin, ALS-, PS II- andpigment synthesis-inhibitor herbicides, such as rimsulfuron,nicosulfuron and mesotrione.

It is therefore envisioned that the same tolerance to certain herbicidespresent in most corn lines can be extended to other crop plants bytransgenic means though the use of the endogenous maize Nsf1 gene andvariants thereof. Listings of maize lines with tolerance or sensitivityto selected SU herbicides are widely available, such as those providedby the USDA, ARS, National Genetic Resources Program. GermplasmResources Information Network—(GRIN). [Online Database] NationalGermplasm Resources Laboratory, Beltsville, Md. [retrieved on Mar. 6,2006]: Retrieved from the internet: <URL:http://www.ars-grin.gov/cgi-bin/npgs/html/dno_eval_acc.pI?89201+153002+21>;and the “Maize Germplasm Lines” listings available from the BucklerLaboratory website [retrieved on Mar. 6, 2006]: Retrieved from theinternet: <URL:http://www.maizegenetics.net/index.php?page=germplasm/lines.html>, andalso in reference articles such as Kang (1993) J. Heredity. 84(3):216-217.

Where appropriate, the polynucleotides may be optimized for increasedexpression in the transformed organism. For example, the polynucleotidescan be synthesized using plant-preferred codons for improved expression.See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (Zea mays),Brassica sp. (e.g., B. napus, B. rapa, B. juncea), alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats (Avena spp.), barley, palm, coconut, castor bean, olive, beans (forexample guar, locust bean, fenugreek, soybean, garden beans, mung beans,lima beans, fava beans), peas (such as cowpeas, field peas, lentils,chickpeas, etc.), vegetables, ornamentals, and conifers.

Other plants of interest for the invention include those which have thepotential for use as biofuel crops, including, but not limited to,prairie grasses such as switchgrass (Panicum virgatum), elephant grass(Pennisetum purpureum), Johnson grass (Sorghum halepense), Miscanthusspp., as well as hybrid poplar and hybrid willow trees.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

The embodiments provide not only a gene for use in transgenicapplications, but sequences and methods that allow the resistance geneto be used as a marker in corn breeding strategies. For example, thegene of the embodiments, or the locus containing it, may be identifiedin a crop line intended to be used for breeding. Breeders wouldgenerally want to avoid using crop lines that are sensitive toherbicides where there is usually natural tolerance. Accordingly, theidentification of the sequence of the Nsf1 gene will help breeders toidentify and avoid creating herbicide-sensitive lines.

Nucleic acid based markers can be developed and applied using manydifferent technologies. Such technologies include, and are not limitedto, Restriction Fragment Length Polymorphism (RFLP), Simple SequenceRepeat (SSR), Random Amplified Polymorphic DNA (RAPD), Cleaved AmplifiedPolymorphic Sequences (CAPS) (Rafalski and Tingey, 1993, Trends inGenetics 9:275-280), Amplified Fragment Length Polymorphism (AFLP) (Voset al., 1995, Nucleic Acids Res. 23:4407-4414), Single NucleotidePolymorphism (SNP) (Brookes, 1999, Gene 234:177-186), SequenceCharacterized Amplified Region (SCAR) (Paran and Michelmore, 1993,Theor. Appl. Genet. 85:985-993), Sequence Tagged Site (STS) (Onozaki etal., 2004, Euphytica 138:255-262), Single Stranded ConformationPolymorphism (SSCP) (Orita et al, 1989, Proc Natl Acad Sci USA86:2766-2770), Inter-Simple Sequence Repeat (ISSR) (Blair et al, 1999,Theor. Appl. Genet. 98:780-792), Inter-Retrotransposon AmplifiedPolymorphism (IRAP), Retrotransposon-Microsatellite AmplifiedPolymorphism (REMAP) (Kalendar et al. (1999) Theor. Appl. Genet.98:704-711) and the like.

As used herein, “locus” shall refer to a genetically defined region of achromosome carrying a gene or, possibly, two or more genes so closelylinked that genetically they behave as a single locus, responsible for aphenotype. A “gene” shall refer to a specific gene within that locus,including its associated regulatory sequences. Thus, the Nsf1 locusrefers to the chromosomal region genetically defined as conferringresistance to at least one herbicide of the ALS-inhibiting,PPO-inhibiting, pigment synthesis-inhibiting, PS II-inhibiting andsynthetic auxin herbicide class. One embodiment of the present inventionis the isolation of the Nsf1 gene and the demonstration that it is thegene responsible for the phenotype conferred by the presence of thelocus. Genetically defined loci are by their nature not as preciselydefined in terms of size as genes, which can be delineated molecularly.

Units, prefixes, and symbols may be denoted in their Si accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges are inclusiveof the numbers defining the range. Amino acids may be referred to hereinby either their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. The above-defined terms are more fullydefined by reference to the specification as a whole.

EXAMPLES

The embodiments of the invention are further defined in the followingExamples, in which all parts and percentages are by weight and degreesare Celsius, unless otherwise stated. It should be understood that theseExamples, while indicating embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthe embodiments of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications to adaptit to various usages and conditions. Thus, various modifications of theembodiments of the invention in addition to those shown and describedherein will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. The disclosure of each reference set forthherein is incorporated by reference in its entirety

Example 1 Identification of the Nsf1 Gene through Positional Cloning

A BC1 population (expected 50% Nsf1/nsf1, 50% nsf1/nsf1) was developedusing the sensitive inbred W703A as the recurrent parent, and either B73or Q66 as the resistant line. Plants were misted with a 2.3 mMnicosulfuron, 0.5% v/v Kinetic surfactant solution at approximately theV3 stage. Both resistant and sensitive parents were also grown andsprayed as controls. In order to avoid falsely classifying a plant whichmay have died due to reasons other than the herbicide application, onlyresistant progeny were sampled and analyzed. A total of 96 resistantplants were used for the initial mapping. This was sufficient to placeNsf1 between markers umc1766 and umc2036, and thus on contig 202 of themaizeB73-based physical map ((Retrieved on Mar. 6, 2006) Retrieved fromthe internet <URL: http://www.gramene.org/Zea _(—)mays/cytoview?contig=ctg202&x=44&y=9>).

Based on BAC-end sequences of a maize Mo17-based contig, flanking CAPS(cleaved amplified polymorphic sequence) markers were identified on BACsof contig 202.

For finer mapping of this interval, a total of 388 resistant plants wereused in the next step. Based on sequencing of subcloned fragments ofBACs in this interval, two flanking CAPs markers were found onoverlapping BACs. Both of these markers had 2/388 recombinants.

Both of these BACs were sequenced and analyzed. Within the 163 kb regionof the 2 BACs flanked by two proprietary markers, P1 and P2, there wereseveral putative genes. For the third round of mapping, a total of 2584resistant plants were used, and markers were developed to separate someof the genes. One marker showed 11/2584 (0.4%) recombinants, helping toeliminate certain genes as being responsible for the resistance. Twoother markers each had 2 (0.08%) recombinants, eliminating yet anothergene. Finally, a marker between two genes had a single recombinant(0.04%), eliminating one of those two genes. Thus it was determinedwhich gene was the gene of interest. The gene, Nsf1, was determined tohave homology to some cytochrome P450 genes known in the art.

Example 2 Analysis of the Nsf1 Gene

Analysis of the Gene 18 (Nsf1) sequence in the B73-derived BAC shows anopen reading frame of 521 amino acids, and containing the conservedheme-binding motif FXXGXXXCXG (SEQ ID NO: 14) found in all cytochromeP450s (FIGS. 1 d and 2 b).

In order to determine if the Nsf1 allele was consistent across maizelines, three corn lines with unknown sensitivity levels to nicosulfuronwere tested to determine their reaction and then evaluate theirsequences. Plants were misted with a 2.3 mM nicosulfuron, 0.5% v/vKinetic surfactant solution at approximately the V3 stage. Both knownresistant and sensitive lines were also grown and sprayed as controls.Results of the testing of the three lines showed that lines Q66 andBlack Mexican Sweet (BMS) were resistant and line A188 was sensitive.

Of these two other resistant lines, Q66 and BMS, also possess this ORF,although Q66 differs from both B73 and BMS by 3 amino acids (FIGS. 2 aand 2 b) These three variant amino acids are marked with bold type andrectangles in FIGS. 2 a and 2 b in the Q66 sequence string to show theirpositions. Analysis of a sensitive line, GA209, shows an insertion of392 bp relative to the resistant lines which results in a frameshift andan open reading frame of only 338 amino acids (FIG. 2 b). A survey ofnumerous North American sensitive lines showed that many of thesensitive lines contain this same insertion of unknown DNA.

Analysis of the sequence from the F2 sensitive line showed that there isonly one nucleotide difference between B73 (SEQ ID NO: 2) and F2 (SEQ IDNO: 22), which changes amino acid 263 from arginine to threonine (FIG. 2b). This single change therefore eliminates the resistance phenotype andvariant sequences with such a change are expected not to retainbiological activity. This change is useful in developing an SNP toassist corn breeders in avoiding the susceptible allele.

Nsf1 is 67% identical to a rice cytochrome P450 which has recently beenreported to control sulfonylurea sensitivity in that plant (AccessionNo: ABC69856, SEQ ID NO: 4).

Genomic sequence from B73 shows a single intron with the expected GTleft border and AG right border. The position of the intron is shown inthe sequence listing in SEQ ID NO: 16.

The cloning of this gene has a number of potential applications. Itcould be used as a selectable marker for transformation in a sensitivetransformable line such as A188 (Ishida et al., (1996) NatureBiotechnology 14:745-750). A transgene designed to suppress the Nsf1gene function would function as a dominant negative selectable marker.Nsf1 could also be used to create transgenic resistance in other plants,such as soybean, which are sensitive to this subclass of sulfonylureas.

Example 3 Testing of Maize Plants for Sensitivity to Nicosulfuron

Three corn lines with unknown sensitivity levels to nicosulfuron weretested to determine their reaction. Plants were misted with a 2.3 mMnicosulfuron, 0.5% v/v Kinetic surfactant solution at approximately theV3 stage. Both known resistant and sensitive lines were also grown andsprayed as controls. Results of the testing of the three lines showedthat lines Q66 and BMS were resistant and line A188 was sensitive.

Example 4 Preparation of Transgenic Soybean Plants

The following stock solutions and media were used for transformation andregeneration of soybean plants:

Stock solutions

-   Sulfate 100× Stock: 37.0 g MgSO₄.7H₂O, 1.69 g MnSO₄.H₂O, 0.86 g    ZnSO₄.7H₂O, 0.0025 g CuSO₄.5H₂O.-   Halides 100× Stock: 30.0 g CaCl₂.2H₂O, 0.083 g KI, 0.0025 g    CoCl₂.6H₂O,-   P, B, Mo 100× Stock: 18.5 g KH₂PO₄, 0.62 g H₃BO₃, 0.025 g    Na₂MoO₄.2H₂O-   Fe EDTA 100× Stock: 3.724 g Na₂EDTA, 2.784 g FeSO₄.7H₂O.-   2,4-D Stock: 10 mg/mL.-   Vitamin B5 1000× Stock: 10.0 g myo-inositol, 0.10 g nicotinic acid,    0.10 g pyridoxine HCl, 1 g thiamine.    Media (per Liter)-   SB196: 10 mL of each of the above stock solutions, 1 mL B5 Vitamin    stock, 0.463 g (NH₄)₂SO₄, 2.83 g KNO₃, 1 mL 2,4-D stock, 1 g    asparagine,10 g sucrose, pH 5.7.-   SB103: 1 pk. Murashige & Skoog salts mixture, 1 mL B5 Vitamin stock,    750 mg MgCl₂ hexahydrate, 60 g maltose, 2 g gelrite, pH 5.7.-   SB166: SB103 supplemented with 5 g per liter activated charcoal.-   SB71-4: Gamborg's B5 salts (Gibco-BRL catalog No. 21153-028), 1 mL    B5 vitamin stock, 30 g sucrose, 5 g TC agar, pH 5.7.

Soybean embryogenic suspension cultures were maintained in 35 mL liquidmedium (SB196) on a rotary shaker (150 rpm) at 28° C. with fluorescentlights providing a 16-hour day/8-hour night cycle. Cultures weresubcultured every 2 weeks by inoculating approximately 35 mg of tissueinto 35 mL of fresh liquid media.

Soybean embryogenic suspension cultures were transformed by particle gunbombardment (see Klein et al. (1987) Nature 327:70-73) using a DuPontBiolistic PDS1000/He instrument.

The recombinant DNA plasmid used to express Nsf1 was on a separaterecombinant DNA plasmid from the selectable marker gene. Bothrecombinant DNA plasmids were co-precipitated onto gold particles asfollows. The DNAs in suspension were added to 50 μL of a 20-60 mg/mL 0.6μm gold particle suspension and then combined with 50 μL CaCl₂ (2.5 M)and 20 μL spermidine (0.1 M). The mixture was pulse vortexed 5 times,spun in a microfuge for 10 seconds, and the supernatant removed. TheDNA-coated particles are then washed once with 150 μL of 100% ethanol,pulse vortexed and spun in a microfuge again, and resuspended in 85 μLof anhydrous ethanol. Five μL of the DNA-coated gold particles are thenloaded on each macrocarrier disk.

Approximately 150 to 250 mg of two-week-old suspension culture wasplaced in an empty 60 mm×15 mm petri plate and the residual liquid isremoved from the tissue using a pipette. The tissue was placed about 3.5inches away from a retaining screen and each plate of tissue wasbombarded once. Membrane rupture pressure was set at 650 psi and thechamber was evacuated to −28 inches of Hg. Eighteen plates werebombarded, and, following bombardment, the tissue from each plate wasdivided between two flasks, placed back into liquid media, and culturedas described above.

Seven days after bombardment, the liquid medium was exchanged with freshSB196 medium supplemented with 50 mg/mL hygromycin. The selective mediumwas refreshed weekly or biweekly. Seven weeks post-bombardment, green,transformed tissue was observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue was removed and inoculatedinto individual flasks to generate new, clonally-propagated, transformedembryogenic suspension cultures. Thus, each new line was treated as anindependent transformation event. These suspensions were then maintainedas suspensions of embryos clustered in an immature developmental stagethrough subculture or were regenerated into whole plants by maturationand germination of individual somatic embryos.

Transformed embryogenic clusters were removed from liquid culture andplaced on solid agar medium (SB166) containing no hormones orantibiotics for one week. Embryos were cultured at 26° C. with mixedfluorescent and incandescent lights on a 16-hour day: 8-hour nightschedule. After one week, the cultures were then transferred to SB103medium and maintained in the same growth conditions for 3 additionalweeks. Prior to transfer from liquid culture to solid medium, tissuefrom selected lines was assayed by PCR for the presence of the chimericgene. Somatic embryos became suitable for germination after 4 weeks andwere then removed from the maturation medium and dried in empty petridishes for one to five days. The dried embryos were then planted inSB71-4 medium and allowed to germinate under the same light andgermination conditions described above. Germinated embryos weretransferred to sterile soil and grown to maturity.

Example 5 T0 and T1 Transgenic Plant Analysis

T0 Testing

Two different constructs comprising the Nsf1 gene were created toexamine herbicide efficacy of the gene when transformed into soybean.The Nsf1 constructs were co-bombarded with a 35S:HYG insert to permitevent selection using hygromycin.

At the V2 to V6 growth stage, a total of 127 T0 plants were sprayed with35 g/ha rimsulfuron. All rimsulfuron treatments were applied with 0.2%w/w nonionic surfactant in a spray volume of 287 L/ha. In addition tothe T0 plants, replications of three different controls wereincluded—two positive and one negative. Individual plants were evaluatedfor herbicide response at ten days after treatment, and assigned avisual response score from 1 to 9 (1=dead plant to 9=no effectobserved). Based upon high tolerance scores to the initial rimsulfuronspray, five T0 events were sprayed with an additional 35 g/harimsulfuron. Plants were rated for visual tolerance using a 1 to 9 scoreat ten days after the second application.

In the T0 generation, 4 of 51 events had improved tolerance compared tothe controls at ten days after treatment with 35 g/ha rimsulfuron. Threeof 51 T0 events had improved level of tolerance after an additionalapplication of 35 g/ha rimsulfuron. Two of these 51 events were advancedto the T1 generation for more extensive herbicide testing.

T1 Testing

Two events from the T0 generation were advanced to the T1 generation foradditional herbicide efficacy testing of the Nsf1 gene. Replicates oftwo controls, as well as T1 plants, were grown in greenhouse experimentsand sprayed with mesotrione at one of two rates (200 g/ha or 50 g/ha),nicosulfuron (70 g/ha), or rimsulfuron (35 g/ha) at the V3 growth stage.All herbicide treatments were applied with 1% w/w modified seed oiladjuvant in a spray volume of 374 L/ha. Plants were rated for herbicideresponse at eight days after application using a 1 to 9 score as used inthe T0 testing.

An expanded herbicide efficacy test was developed in a second T1 plantexperiment for the same two events advanced from the T0 generation. Atthe V3 growth stage, plants were sprayed with different treatments ofherbicides that would typically cause substantial crop injury whenapplied to commodity soybean at the rates examined. All herbicidetreatments were applied in a spray volume of 287 L/ha. Isoxaflutole (140g/ha), topramezone (140 g/ha), and sulcotrione (140 g/ha) were appliedwith 1% w/w modified seed oil adjuvant. Diuron treatments (560 g/ha)were applied with 1% w/w petroleum crop oil adjuvant. Acifluorfen (4480g/ha), sulfentrazone (140 g/ha), flumioxazin (140 g/ha), and dicamba(280 g/ha) were applied with 0.25% w/w nonionic surfactant. Rimsulfuron(35 g/ha) treatments were applied with 0.5% w/w basic blend adjuvant. Ateight and fifteen days after treatment, plants were rated visually forcrop injury using a 0 to 100 scale (0=no injury to 100=dead plant).Since the T1 events were segregating, only the plants with the bestoverall scores were selected, corresponding to the 75% that would beexpected to possess the transgene.

One of the two events had significantly better tolerance compared to thecontrols at 8 DAT and 15 DAT after application of acifluorfen, dicamba,diuron, flumioxazin, isoxaflutole, mesotrione, rimsulfuron, sulcotrione,sulfentrazone, and topramezone treatments. The second event hadsignificantly better tolerance compared to the controls at 15 DAT afterapplication of acifluorfen, dicamba, isoxaflutole, mesotrione,rimsulfuron, sulcotrione, sulfentrazone, and topramezone treatments.Although the exact expression level of the Nsf1 gene in the eventstested was not determined, transgenic soybean plants comprising themaize Nsf1 gene displayed better tolerance to a range of differentherbicides when compared directly to control plants.

1. An isolated polynucleotide comprising: (a) a nucleotide sequenceencoding a polypeptide capable of conferring resistance to at least twoherbicides, wherein each herbicide is a member of a different class ofherbicides selected from the group consisting of: (i) the ALS-inhibitingclass; (ii) the pigment synthesis-inhibiting class; (iii) thePPO-inhibiting class; (iv) the PS II-inhibiting class; and (v) thesynthetic auxin class wherein the polypeptide has an amino acid sequenceof at least 85% identity, when compared to SEQ ID NO:1 based on theNeedleman-Wunsch alignment algorithm, or (b) a complement of thenucleotide sequence, wherein the complement and the nucleotide sequenceconsist of the same number of nucleotides and are 100% complementary. 2.The polynucleotide of claim 1, wherein the amino acid sequence of thepolypeptide and the amino acid sequence of SEQ ID NO: 1 have at least90% identity based on the Needleman-Wunsch alignment algorithm.
 3. Thepolynucleotide of claim 1, wherein the amino acid sequence of thepolypeptide and the amino acid sequence of SEQ ID NO: 1 have at least95% identity based on the Needleman-Wunsch alignment algorithm.
 4. Thepolynucleotide of claim 1, wherein the nucleotide sequence comprises SEQID NO:
 1. 5. A vector comprising the polynucleotide of claim
 1. 6. Arecombinant DNA construct comprising the polynucleotide of claim 1operably linked to at least one regulatory sequence.
 7. A plant cellcomprising the recombinant DNA construct of claim
 6. 8. A plantcomprising the recombinant DNA construct of claim
 6. 9. A seedcomprising the recombinant DNA construct of claim
 6. 10. The plant ofclaim 8, wherein said plant is a monocot.
 11. The plant of claim 10,wherein said monocot is selected from the group consisting of maize,wheat, barley, oats, switchgrass, sorghum, and rice.
 12. The plant ofclaim 8, wherein said plant is a dicot.
 13. The plant of claim 12,wherein said dicot is selected from the group consisting of soybean,canola, potato, cotton, and sunflower.
 14. The plant of claim 8, whereinsaid plant further comprises a gene encoding a polypeptide withglyphosate N-acetyltransferase activity.